Method for inhibiting bone loss using 6-hydroxy-2-(4-hydroxyphenyl)-benzo[B][2-(piperidin-1-yl)ethoxyphenylimethanone hydrochloride

ABSTRACT

The current invention provides a method useful for inhibiting the loss of bone using 6-hydroxy-2-(4-hydroxyphenyl)-benzo(B)thien-3-yl-4[2-(piperidin-1-ethoxyphenol]methanone hydrochloride.

This application is a division, of application Ser. No. 08/180,522 filedJan. 12, 1994, now U.S. Pat. No. 5,393,763, which is a continuation ofapplication Ser. No. 07/920,933 filed Jul. 28, 1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the discovery that a group of2-phenyl-3-aroylbenzothiophenes is useful in the prevention of boneloss.

The mechanism of bone loss is not well understood, but in practicaleffect, the disorder arises from an imbalance in the formation of newhealthy bone and the resorption of old bone, skewed toward a net loss ofbone tissue. This bone loss includes a decrease in both mineral contentand protein matrix components of the bone, and leads to an increasedfracture rate of, predominantly, femoral bones and bones in the forearmand vertebrae. These fractures, in turn, lead to an increase in generalmorbidity, a marked loss of stature and mobility, and, in many cases, anincrease in mortality resulting from complications.

Bone loss occurs in a wide range of subjects, including post-menopausalwomen, patients who have undergone hysterectomy, patients who areundergoing or have undergone long-term administration ofcorticosteroids, patients suffering from Cushing's syndrome, andpatients having gonadal dysgensis.

Unchecked, bone loss can lead to osteoporosis, a major debilitatingdisease whose prominent feature is the loss of bone mass (decreaseddensity and enlargement of bone spaces) without a reduction in bonevolume, producing porosity and fragility.

One of the most common types of osteoporosis is found in post-menopausalwomen affecting an estimated 20 to 25 million women in the United Statesalone. A significant feature of post-menopausal osteoporosis is thelarge and rapid loss of bone mass due to the cessation of estrogenproduction by the ovaries. Indeed, data clearly support the ability ofestrogens to limit the progression of osteoporotic bone loss, andestrogen replacement is a recognized treatment for post-menopausalosteoporosis in the United States and many other countries. However,although estrogens have beneficial effects on bone, given even at verylow levels, long-term estrogen therapy has been implicated in a varietyof disorders, including an increase in the risk of uterine and breastcancer, causing many women to avoid this treatment. Recently suggestedtherapeutic regimens, which seek to lessen the cancer risk, such asadministering combinations of progestogen and estrogen, cause thepatient to experience regular withdrawal bleeding, which is unacceptableto most older women. Concerns over the significant undesirable effectsassociated with estrogen therapy, and the limited ability of estrogensto reverse existing bone loss, support the need to develop alternativetherapy for bone loss that generates the desirable effects on bone butdoes not cause undesirable effects.

Attempts to fill this need by the use of compounds commonly known asantiestrogens, which interact with the estrogen receptor, have hadlimited success, perhaps due to the fact that these compounds generallydisplay a mixed agonist/antagonist effect. That is, although thesecompounds can antagonize estrogen interaction with the receptor, thecompounds themselves may cause estrogenic responses in those tissueshaving estrogen receptors. Therefore, some antiestrogens are subject tothe same adverse effects associated with estrogen therapy.

The current invention provides methods for inhibiting the loss of bonewithout the associated adverse effects of estrogen therapy, and thusserves as an effective and acceptable treatment for osteoporosis.

The 2-phenyl-3-aroylbenzothiophene compounds that are the activecomponent in the formulations and methods of this invention were firstdeveloped by C. David Jones and Tulio Suarez as anti-fertility agents(see U.S. Pat. No. 4,133,814, issued Jan. 9, 1979). Certain compounds inthe group were found to be useful in suppressing the growth of mammarytumors.

Jones later found a group of related compounds to be useful forantiestrogen and antiandrogen therapy, especially in the treatment ofmammary and prostatic tumors (see U.S. Pat. No. 4,418,068, issued Nov.29, 1983). One of these compounds, the compound of formula I wherein Xis a bond, R and R¹ are hydroxyl, and R² is a piperidino ring, wasclinically tested for a brief time for the treatment of breast cancer.That compound is called raloxifene, formerly keoxifene.

SUMMARY OF THE INVENTION

This invention provides new methods for the treatment of bone losscomprising administering to a human in need of treatment an effectiveamount of a compound of formula I ##STR1## wherein X is a bond, CH₂, orCH₂ CH₂ ;

R and R¹, independently, are hydrogen, hydroxyl, C₁ -C₆ -alkoxy, C₁ -C₆-acyloxy, C₁ -C₆ -alkoxy-C₂ -C₆ -acyloxy, R³ -substituted aryloxy, R³-substituted aroyloxy, R⁴ -substituted carbonyloxy, chloro, or bromo;

R² is a heterocyclic ring selected from the group consisting ofpyrrolidino, piperidino, or hexamethyleneimino;

R³ is C₁ -C₃ -alkyl, C₁ -C₃ -alkoxy, hydrogen, or halo; and

R⁴ is C₁ -C₆ -alkoxy or aryloxy; or

a pharmaceutically acceptable salt thereof.

The invention also provides pharmaceutical formulations for inhibitingbone loss comprising a compound of formula I, wherein R, R1, R2, and Xare as defined above in an amount that increases or retains bonedensity, together with a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns the discovery that a group of2-phenyl-3-aroylbenzothiophenes (benzothiophenes) of formula I areuseful in the treatment of osteoporosis. The benzothiophenes of formulaI inhibit the loss of bone that results from a lack of endogenousestrogen such as occurs in women following cessation of menstruation dueto natural, surgical, or other processes. The reduction of bone densityand mass that more rarely occurs in men is also tied to the loss ofhormonal regulation and is therefore also a target for therapy accordingto the methods of the current invention.

The benzothiophenes of formula I are a series of nonsteroidal compoundsthat exhibit high affinity for conventional estrogen receptors inprimary sex target tissues. However, they elicit minimal estrogenicresponses in those tissues, and actually serve as potent antagonists ofnatural estrogens such as estradiol. In contrast to the report ofFeldmann, S. et al., "Antiestrogen and antiandrogen administrationreduce bone mass in the rat", Bone and Mineral, 7:245 (1989), thebenzothiophenes of formula I are able to antagonize classical estrogenicresponses in primary sex target tissues without significantly reducingbone density when given to intact or estrogen treated animals, and theyprevent bone loss in estrogen deficient animals. This dichotomyindicates selective agonist/antagonist actions on specific target cellswhich would appear to be highly desirable in treatment of the menopausalsyndrome. Accordingly, the real benefit of the current discovery is thatthe benzothiophenes of formula I inhibit the loss of bone but do notelicit significant estrogenic responses in the primary sex targettissues. Thus, the current invention provides a method of inhibitingbone loss comprising administering to a human in need of treatment anamount of a compound of formula I that inhibits bone loss but does notsignificantly affect the primary sex target tissues. This combination offeatures allows for long-term treatment of the chronic ailment with adiminished risk of developing the undesirable effects of customaryestrogen replacement therapy.

The biological action of the benzothiophenes of formula I is complex andmay be unrelated to the detectable presence of the parent compound inthe blood. Following oral administration of a preferred benzothiopheneof this invention, raloxifene (raloxifene hydrochloride), to humansubjects in the clinic, the parent compound was not detected in theserum of those subjects. It was determined that following oraladministration, the compound was extensively conjugated to theglucuronidated form and cleared quickly from the bloodstream. Althoughno biological endpoints were measured in the human recipients, there wasconcern that the compound was not bioavailable.

Experiments were undertaken to address the bioavailability issue inlaboratory animals where biological activity could be assessed. Theanimal studies indicated that raloxifene was maximally active ininhibiting both uterine uptake of tritiated-estradiol and the normaluterotrophic response to estradiol even under conditions whereraloxifene was extensively conjugated in the plasma of the animals.Moreover, the conjugate, isolated from the urine of human subjectstreated with raloxifene, displayed significantantiestrogenic/antiuterotrophic activity when administered intravenouslyto rats, and inhibited the interaction of tritiatedestradiol with ratuterine estrogen receptors in a manner similar to the parent compound.These studies suggested the conjugated compound may have been convertedto the parental form at the site of action, presumably by the action ofβ-glucuronidase. Such conversion may contribute to the activity of thecompound. β-Glucuronidase is fairly ubiquitous and is thought to beactive in the resorption process of bone remodeling, and wouldpresumably be available for converting the conjugated compound to theparental form if required for activity. Therefore, conjugation of thebenzothiophenes of formula I is not considered to be necessarilydetrimental to their bioavailability as an inhibitor of bone loss.

Thus, the method of treatment provided by this invention is practiced byadministering to a human in need of inhibition of bone loss, a dose of acompound of formula I or a pharmaceutically acceptable salt thereof,that is effective to inhibit bone loss. A particular benefit of thismethod is that it avoids potentially harmful and unacceptable estrogenicside effects. The inhibition of bone loss contemplated by the presentmethod includes both medical therapeutic and/or prophylactic treatment,as appropriate.

The method also includes the administration of a compound of formula Igiven in combination with estrogen. The term estrogen as used hereinrefers to any compound which approximates the spectrum of activities ofthe naturally acting molecule which is commonly believed to be17β-estradiol. Examples of such compounds include estriol, estrone,ethynyl estradiol, Premarin (a commercial preparation of conjugatedestrogens isolated from natural sources--Ayerst), and the like. Again,due to the selective agonist/antagonist properties of the compounds offormula I, this combination provides for the full benefits of estrogentherapy without the concomitant adverse effects associated with estrogentherapy alone.

The general chemical terms used in the description of a compound offormula I have their usual meanings. For example, the term "C₁ -C₃-alkyl" includes such groups as methyl, ethyl, propyl, and isopropyl.

The term "C₁ -C₆ -alkoxy" includes such groups as methoxy, ethoxy,propoxy, butoxy, pentyloxy, and hexyloxy and also includes branchedchain structures such as, for example, isopropoxy and isobutoxy.

The term "C₁ -C₆ -acyloxy" includes methanoyloxy, ethanoyloxy,propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy, and the like andalso includes branched chain structures such as, for example,2,2-dimethylpropanoyloxy, and 3,3-dimethylbutanoyloxy.

The term "C₁ -C₆ -alkoxy-C₂ -C₆ -acyloxy" contemplates, for example,methoxyethanoyloxy, methoxypropanoyloxy, methoxybutanoyloxy,methoxypentanoyloxy, methoxyhexanoyloxy, ethoxyethanoyloxy,ethoxypropanoyloxy, ethoxybutanoyloxy, ethoxypentanoyloxy,ethoxyhexanoyloxy, propoxyethanoyloxy, propoxypropanoyloxy,propoxybutanoyloxy, and the like.

It should also be understood that as used herein, references to alkyland alkoxy structures also include cycloalkyl and cycloalkoxy groupswhere the number of carbons within the structure is at least 3.

The terms "R³ -substituted aryloxy" and "R³ -substituted aroyloxy"include such groups as phenyloxy, thienyloxy, furyloxy, naphthyloxy,benzoyloxy, thienoyloxy, furoyloxy, naphthoyloxy, and the like, wherethe R³ substitution group may be hydrogen, hydroxyl, C₁ -C₃ -alkyl, C₁-C₃ -alkoxy, or halo.

The term "R⁴ -substituted carbonyloxy, where the R⁴ substitution groupmay be C₁ -C₆ -alkoxy or aryloxy, includes carbonate structures such asmethoxycarbonyloxy ethoxycarbonyloxy, propoxycarbonyloxy,butoxycarbonyloxy, pentyloxycarbonyloxy, hexyloxycarbonyloxy,phenyloxycarbonyloxy, thienyloxycarbonyloxy, furyloxycarbonyloxy, andnaphthyloxycarbonyloxy.

Preferred methods of this invention comprise the use of compounds offormula I wherein R and R¹ are other than hydrogen, alkoxy, aryloxy,chloro, or bromo and therefore represent ester and carbonateconfigurations. Other preferred methods include the use of formula Icompounds wherein R and R¹ are the same as one another. Certain R²groups also demonstrate preferable characteristics when used in themethods of this invention. For example, preferred methods of thisinvention include the use of formula I compounds wherein R² ispiperidino or pyrrolidino, especially piperidino. A further preferredsubgroup of the preferred piperidino and pyrrolidino compounds includecompounds wherein R and R¹ are other than hydrogen and, in particular,those wherein R and R¹ are hydroxyl.

All of the compounds used in the methods of the current invention can bemade according to established procedures, such as those detailed in U.S.Pat. Nos. 4,133,814 and 4,418,068. In general, the process starts with abenzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxyphenyl)group. The starting compound is protected, alkylated, and deprotected toform the formula I compounds wherein R and R¹ are both hydroxy. Theformula I compounds that are ethers, esters, and carbonates may then beformed if desired. Examples of the preparation of such compounds areprovided in the U.S. patents discussed above. Specific preparations ofyet other derivatized compounds useful in the current invention areoutlined in the Preparations sections below. Modifications to the abovemethods may be necessary to accommodate reactive functionalities ofparticular substituents. Such modifications would be both apparent to,and readily ascertained by, those skilled in the art.

The compounds used in the methods of this invention formpharmaceutically acceptable acid and base addition salts with a widevariety of organic and inorganic acids and bases and include thephysiologically acceptable salts which are often used in pharmaceuticalchemistry. Such salts are also part of this invention. Typical inorganicacids used to form such salts include hydrochloric, hydrobromic,hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, phenylacetate, trifluoroacetate, acrylate,ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methane-sulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartarate, and the like.

In addition, some of the formula I compounds may form solvates withwater or organic solvents such as ethanol. These solvates are alsocontemplated for use in the methods of this invention.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates andbicarbonates, as well as aliphatic and aromatic amines, aliphaticdiamines and hydroxy alkylamines. Bases especially useful in thepreparation of addition salts include ammonium hydroxide, potassiumcarbonate, sodium bicarbonate, calcium hydroxide, methylamine,diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

The current invention also provides useful pharmaceutical formulationsfor inhibiting bone loss comprising a formula I compound plus one ormore pharmaceutically acceptable excipients. Pharmaceutical formulationscan be prepared by procedures known in the art. For example, thecompounds can be formulated with common excipients, diluents, orcarriers, and formed into tablets, capsules, suspensions, powders, andthe like. Examples of excipients, diluents, and carriers that aresuitable for such formulations include the following: fillers andextenders such as starch, sugars, mannitol, and silicic derivatives;binding agents such as carboxymethyl cellulose and other cellulosederivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizingagents such as glycerol; disintegrating agents such as agaragar, calciumcarbonate, and sodium bicarbonate; agents for retarding dissolution suchas paraffin; resorption accelerators such as quaternary ammoniumcompounds; surface active agents such as cetyl alcohol, glycerolmonostearate; adsorptive carriers such as kaolin and bentonite; andlubricants such as talc, calcium and magnesium stearate, and solidpolyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

The particular dosage of a compound of formula I required to treat orinhibit bone loss according to this invention will depend upon theseverity of the disease, its route of administration, and relatedfactors that will be decided by the attending physician. Generally,accepted and effective doses will be from about 0.1 to about 1000 mg,and more typically from about 200 to about 600 mg. Such dosages will beadministered to a subject in need of treatment from once to about threetimes each day, or more often as needed to effectively inhibit the boneloss process.

It is usually preferred to administer a compound of formula I in theform of an acid addition salt, as is customary in the administration ofpharmaceuticals bearing a basic group such as the piperidino ring. It isalso advantageous to administer such a compound by the oral route to anaging human (e.g. a post-menopausal female or a male showing evidence ofbone loss by X-ray analysis). For such purposes the following oraldosage forms are available.

FORMULATIONS

In the formulations which follow, "Active ingredient" means a compoundof formula I.

    ______________________________________                                        Formulation 1: Gelatin Capsules                                               Hard gelatin capsules are prepared using the following:                       Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.1-1000                                                    Starch, NF        0-650                                                       Starch flowable powder                                                                          0-650                                                       Silicone fluid 350 centistokes                                                                  0-15                                                        ______________________________________                                    

The ingredients are blended, passed through a No. 45 mesh U.S. sieve,and filled into hard gelatin capsules.

Examples of specific capsule formulations containing raloxifene thathave been made include those shown below:

    ______________________________________                                        Formulation 2: Raloxifene capsule                                             Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        1                                                           Starch, NF        112                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

    ______________________________________                                        Formulation 3: Raloxifene capsule                                             Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        5                                                           Starch, NF        108                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

    ______________________________________                                        Formulation 4: Raloxifene capsule                                             Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        10                                                          Starch, NF        103                                                         Starch flowable powder                                                                          225.3                                                       Silicone fluid 350 centistokes                                                                  1.7                                                         ______________________________________                                    

    ______________________________________                                        Formulation 5: Raloxifene capsule                                             Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Raloxifene        50                                                          Starch, NF        150                                                         Starch flowable powder                                                                          397                                                         Silicone fluid 350 centistokes                                                                  3.0                                                         ______________________________________                                    

The specific formulations above may be changed in compliance with thereasonable variations provided.

A tablet formulation is prepared using the ingredients below:

    ______________________________________                                        Formulation 6: Tablets                                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                              0.1-1000                                                     Cellulose, microcrystalline                                                                    0-650                                                        Silicon dioxide, fumed                                                                         0-650                                                        Stearate acid    0-15                                                         ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.1-1000 mg of active ingredientare made up as follows:

    ______________________________________                                        Formulation 7: Tablets                                                        Ingredient          Quantity (mg/tablet)                                      ______________________________________                                        Active ingredient   0.1-1000                                                  Starch              45                                                        Cellulose, microcrystalline                                                                       35                                                        Polyvinylpyrrolidone                                                                              4                                                         (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                    4.5                                                       Magnesium stearate  0.5                                                       Talc                1                                                         ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose aremade as follows:

    ______________________________________                                        Formulation 8: Suspensions                                                    Ingredient          Quantity (mg/5 ml)                                        ______________________________________                                        Active ingredient   0.1-1000   mg                                             Sodium carboxymethyl cellulose                                                                    50         mg                                             Syrup               1.25       mg                                             Benzoic acid solution                                                                             0.10       mL                                             Flavor              q.v.                                                      Color               q.v.                                                      Purified water to   5          mL                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

Illustrative compounds that can be used in the formulations and methodsof this invention are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Compound                                                                      No.   X    R and R.sup.1  R.sup.2   Form                                      __________________________________________________________________________    1     bond                                                                                ##STR2##      piperidino                                                                              base                                      2     bond                                                                                ##STR3##      piperidino                                                                              HCl                                       3     bond                                                                                ##STR4##      piperidino                                                                              base                                      4     bond                                                                                ##STR5##      piperidino                                                                              HCl                                       5     bond OC(O)CH.sub.2 CH.sub.2 CH.sub.3                                                              piperidino                                                                              base                                      6     bond OC(O)CH.sub.2 CH.sub.2 CH.sub.3                                                              piperidino                                                                              HCl                                       7     bond OC(O)C(CH.sub.3).sub.3                                                                       piperidino                                                                              base                                      8     bond OC(O)C(CH.sub.3).sub.3                                                                       piperidino                                                                              HCl                                       9     bond OC(O)CH.sub.2 C(CH.sub.3).sub.3                                                              piperidino                                                                              base                                      10    bond OC(O)CH.sub.2 C(CH.sub.3).sub.3                                                              piperidino                                                                              HCl                                       11    bond                                                                                ##STR6##      piperidino                                                                              HCl                                       12    bond                                                                                ##STR7##      piperidino                                                                              base                                      13    bond OC(O)OCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.3                                                    piperidino                                                                              base                                      14    bond OC(O)OCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.3                                                    piperidino                                                                              HCl                                       15    bond                                                                                ##STR8##      piperidino                                                                              base                                      16    bond                                                                                ##STR9##      piperidino                                                                              HCl                                       17    bond                                                                                ##STR10##     piperidino                                                                              base                                      18    bond OC(O)CH.sub.2 CH.sub.2 OCH.sub.3                                                             piperidino                                                                              base                                      19    bond OC(O)CH.sub.2 CH.sub.2 OCH.sub.3                                                             piperidino                                                                              HCl                                       20    bond OH             piperidino                                                                              base                                      21    bond OH             piperidino                                                                              HCl                                       22    bond H              piperidino                                                                              base                                      23    CH.sub.2                                                                           OH             piperidino                                                                              HCl                                       24    CH.sub.2 CH.sub.2                                                                  OH             piperidino                                                                              HCl                                       25    CH.sub.2                                                                           H              piperidino                                                                              HCl                                       26    bond OH             pyrrolodino                                                                             base                                      27    bond OH             pyrrolodino                                                                             HCl                                       28    CH.sub.2                                                                           OH             pyrrolodino                                                                             HCl                                       29    CH.sub.2 CH.sub.2                                                                  OH             pyrrolodino                                                                             HCl                                       30    bond H              pyrrolodino                                                                             HCl                                       31    bond OH             hexamethyleneimino                                                                      HCl                                       32    CH.sub.2                                                                           OH             hexamethyleneimino                                                                      HCl                                       33    CH.sub.2 CH.sub.2                                                                  OH             hexamethyleneimino                                                                      HCl                                       34    bond OCH.sub.3      piperidino                                                                              HCl                                       __________________________________________________________________________

In the following Preparations, the compound numbers correspond to thosegiven in Table 1

Preparation 1

Preparation of Compound 1:6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Raloxifene,6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl-4-[2-(piperidin-1-yl)ethoxyphenyl]methanonehydrochloride, (5.1 g, 10 mmol) was suspended in 250 mL of drytetrahydrofuran (THF) and 7.1 g (70 mmol) of triethylamine, andapproximately 10 mg of 4-(N,N-dimethylamino)pyridine were added. Thesuspension was cooled in an ice bath and placed under an atmosphere ofnitrogen. 4-Fluorobenzoyl chloride (4.75 g, 30 mmol), dissolved in 20 mLof dry THF, was slowly added over a twenty minute period. The reactionmixture was stirred and allowed to slowly warm to room temperature overa period of eighteen hours. It was then filtered, and the filtrate wasevaporated to a gum in vacuo. The crude product thus obtained wasdissolved in a small volume of chloroform and chromotagraphed (HPLC) ona silica gel column eluted with a linear gradient of solvent, startingwith chloroform and ending with a mixture of chloroform-methanol (19:1(v/v)). The fractions containing the desired product as determined bythin layer chromatography (silica, chloroform-methanol (9:1)) werecombined and evaporated to a gum. The final product was crystallizedfrom ether to give 3.21 g of compound 1.

PMR: consistent with the structure FDMS: m/e=717 M+ Elemental Analysisfor C₄₂ H₃₃ F₂ NO₆ S: Theor: C, 70.29; H, 4.60; N, 1.95 Found: C, 70.05;H, 4.60; N, 1.89 Mol. Wt.: 717

Preparation 2

Preparation of Compound 2:6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)-phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]-phenyl]methanonehydrochloride.

Compound 1 (5.15 g, 7.18 mmol) was dissolved in 25 mL THF, and 150 mLether was added. Dry HCl gas was bubbled into the solution, and a whitegummy precipitate formed. The liquid was removed by decanting, and theresidue was crystallized from ethyl acetate with a small amount ofethanol added to effect solution. The product was filtered, washed withether, and dried to give 4.41 g of Compound 2 as a white powder.

PMR: consistent with the structure Elemental Analysis for C₄₂ H₃₄ ClF₂NO₆ S: Theor: C, 66.88; H, 4.54; N, 1.86 Found: C, 66.59, H, 4.39; N,1.60 Mol. Wt.: 753.5

Preparation 3

Preparation of Compound 3:6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

The title compound was prepared using procedures analogous to those inPreparation 1, but using cyclopropylcarbonyl chloride, except that theproduct was not crystallized. Yield=2.27 g.

PMR: consistent with the structure FDMS: m/e=610 M⁺

Preparation 4

Preparation of Compound 4:6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 4 was prepared from Compound 3 as described in Preparation 2.

Preparation 5

Preparation of Compound 5:6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 5 was prepared using the method of Preparation 1, but startingwith n-butanoyl chloride, to give 4.12 g of final product as an oil.

PMR: consistent with the structure FDMS: m/e=614 (M⁺¹)

Preparation 6

Preparation of Compound 6:6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 5 (4.12 g) was dissolved in ethyl acetate (50 mL), and asolution of HCl in ether was added until the precipitation stopped. Theliquid was decanted off, and the white, gummy residue was trituratedwith diethyl ether and filtered. The residue was dried to give 1.33 g ofCompound 6.

PMR: consistent with the structure Elemental Analysis of for C₃₆ H₄₀ClNO₆ S: Theor.: C, 66.50; H, 6.20; N, 2.15 Found: C, 66.30; H, 6.28; N,1.98 Mol. Wt.: 650.24

Preparation 7

Preparation of Compound 7:6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 7 was prepared using the procedure of Preparation 1, but using2,2-dimethylpropanoyl chloride.

Preparation 8

Preparation of Compound 8:6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(piperidin-1yl)ethoxy]phenyl]methanonehydrochloride.

Compound 8 was prepared from Compound 7, as described in Preparation 2.

FDMS: m/e=641 (M-HCl-1) Elemental Analysis of C₃₈ H₄₄ ClNO₆ S: Theor.:C, 67.2 9; H, 6.54; N, 2.07 Found: C, 67.02; H, 6.54; N, 1.90 Mol. Wt.:678.29

Preparation 9

Preparation of Compound 9:6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 9 was prepared using the procedures of Preparation 1, but with3,3-dimethylbutanoyl chloride.

Preparation 10

Preparation of Compound 10:6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 10 was prepared from Compound 9 as described in Preparation 2.

FDMS: m/e=669 (M-HCl-1) Elemental Analysis of C₄₀ H₄₈ ClNO₆ S: Theor.:C, 68.02; H, 6.85; N, 1.98 Found: C, 67.75; H, 6.83; N, 2.04 Mol. Wt.:706.35

Preparation 11

Preparation of Compound 11:6-(4-Methylbenzoyloxy)-2-[4-(4-methylbenzoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 11 was prepared from the free base using a procedure similar tothat of Preparation 2.

FDMS.: m/e=710 (M-HCl-1) Elemental Analysis of C₄₄ H₄₀ ClNO₆ S: Theor.:C, 70.81; H, 5.39; N, 1.88 Found: C, 71.10; H, 5.39; N, 1.94 Mol. Wt.:746.33

Preparation 12

Preparation of Compound 12:6-Benzoyloxy-2-[4-benzoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]-phenyl]methanone.

Compound 12 was prepared from the appropriate acid chloride as describedin Preparation 1.

FDMS: m/e=682 (M+1) Elemental Analysis of C₄₂ H₃₅ NO₆ S: Calc: C, 73.80;H, 5.14; N, 2.05 Found: C, 73.27; H, 5.27; N, 1.94 Mol. Wt.: 681.8

Preparation 13

Preparation of Compound 13: 6-(n-Butoxyoyloxy)-2-[4(n-butoxyoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 13 was prepared in a manner analogous to that described inPreparation 1, except that n-butylchloroformate was used in place of theacid chloride. Yield=6.13 g in form of oil.

PMR: consistent with structure FDMS: m/e=674 (M+1)

Preparation 14

Preparation of Compound 14:6-(n-Butoxycarbonyloxy)-2-[4(n-butoxycarbonyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 13 was converted to the hydrochloride salt in a manneranalogous to that described in Preparation 6.

PMR: consistent with structure Elemental Analysis of C₃₈ H₄₄ ClNO₈ S:Calc: C, 64.26; H, 6.24; N, 1.97 Found: C, 63.97; H, 6.34; N, 1.98 Mol.Wt.: 710.29

Preparation 15

Preparation of Compound 15:6-(Phenyloxycarbonyloxy)-2-[4(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

This compound was prepared in a manner analogous to that described inPreparation 13, but using the appropriate acyl ester. Yield=3.59 g offinal product as a tan amorphous powder.

PMR: consistent with structure FDMS: m/e=713 (M+)

Preparation 16

Preparation of Compound 16:6-(Phenyloxycarbonyloxy)-2-[4(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 15 was converted to the hydrochloride salt in a manneranalogous to that described in Preparation 6.

PMR: consistent with structure Elemental Analysis of C₃₈ H₄₄ ClNO₈ S:Calc: C, 67.24; H, 4.84; N, 1.87 Found: C, 66.94; H, 4.96; N, 1.84 Mol.Wt.: 750.27

Preparation 17

Preparation of Compound 17:6-(Naphthoyloxy)-2-[4(1-naphthoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 17 was prepared as described in Preparation 1 using theappropriate acid halide. Yield=3.5 g of a white amorphous powder

PMR: consistent with structure FDMS: m/e=781 (M+) Elemental Analysis ofC₅₀ H₃₉ NO₆ S: Calc: C, 76.80; H, 5.03; N, 1.79 Found: C, 76.53; H,5.20; N, 1.53 Mol. Wt.: 781.94

Preparation 18

Preparation of Compound 18:6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone.

Compound 18 was prepared as described in Preparation 1 using theappropriate acid halide. Yield=3.61 g of a gummy solid.

PMR: consistent with structure FDMS: m/e=618 (M+1)

Preparation 19

Preparation of Compound 19:6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanonehydrochloride.

Compound 19 was prepared from 3.5 g of Compound 18 as described inPreparation 2. Yield=1.65 g of amorphous white powder.

PMR: consistent with structure FDMS: m/e=618 (M+1) Elemental Analysis ofC₃₄ H₃₆ NO₈ S: Calc: C, 62.43; H, 5.55; N, 2.14 Found: C, 62.23; H,5.63; N, 2.15

The following nonlimiting examples illustrate the methods andformulations of this invention.

EXAMPLE 1

In the examples illustrating the methods, a model of post-menopausalosteoporosis was used in which effects of different treatments uponfemur density were determined.

Seventy-five day old female Sprague Dawley rats (weight range of 225 to275 g) were obtained from Charles River Laboratories (Portage, Mi.).They were housed in groups of 3 and had ad libitum access to food(calcium content approximately 1%) and water. Room temperature wasmaintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. Thephotoperiod in the room was 12 hours light and 12 hours dark.

One week after arrival, the rats underwent bilateral ovariectomy underanesthesia (44 mg/kg Ketamine and 5 mg/kg Xylazine (Butler,Indianapolis, Ind.) administered intramuscularly). Treatment withvehicle, estrogen, or a compound of formula I was initiated on the dayof surgery following recovery from anesthesia. Oral dosage was by gavagein 0.5 mL of 1% carboxymethylcellulose (CMC). Body weight was determinedat the time of surgery and weekly thereafter and the dosage was adjustedwith changes in body weight. Vehicle or estrogen treated ovariectomized(ovex) rats and non-ovariectomized (intact) rats were evaluated inparallel with each experimental group to serve as negative and positivecontrols.

The rats were treated daily for 35 days (6 rats per treatment group) andsacrificed by decapitation on the 36th day. The 35 day time period wassufficient to allow maximal reduction in bone density, measured asdescribed herein. At the time of sacrifice, the uteri were removed,dissected free of extraneous tissue, and the fluid contents wereexpelled before determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight wasroutinely reduced about 75% in response to ovariectomy. The uteri werethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

The right femurs were excised and scanned at the distal metaphysis 1 mmfrom the patellar groove with single photon absorptiometry. Results ofthe densitometer measurements represent a calculation of bone density asa function of the bone mineral content and bone width.

INFLUENCE OF RALOXIFENE ON BONE DENSITY

The results of control treatments from five separate experiments areaccumulated in Table 2. In summary, ovariectomy of the rats caused areduction in femur density of about 25% as compared to intact vehicletreated controls. Estrogen, administered in the orally active form ofethynyl estradiol (EE₂), prevented this loss of bone in a dose dependentmanner, but it also exerted a stimulatory action on the uterus resultingin uterine weights approaching that of an intact rat when administeredat 100 μg/kg. Results are reported as the mean of measurements fromthirty rats ± the standard error of the mean.

In these studies, raloxifene also prevented bone loss in a dosedependent manner; however, only minimal increase of uterine weight overthe ovariectomized controls was present in these animals. The results offive assays using raloxifene are combined in Table 3. Accordingly, eachpoint reflects the responses of thirty rats and depicts a typical doseresponse curve for raloxifene in this model. Results are reported as themean ± the standard error of the mean.

                  TABLE 2                                                         ______________________________________                                                      Bone Density                                                                           Uterine Weight                                                       (mg/cm/cm)                                                                             (mg)                                                   ______________________________________                                        Ovariectomy control                                                                           170 ± 3 127 ± 5                                         (0.5 mL CMC oral)                                                             Intact control  220 ± 4 545 ± 19                                        (0.5 mL CMC oral)                                                             EE.sub.2 100 μg/kg, oral                                                                   210 ± 4 490 ± 11                                        ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                       Bone Density                                                                           Uterine Weight                                                       (mg/cm/cm)                                                                             (mg)                                                  ______________________________________                                        Ovariectomy control                                                                            171 ± 3 127 ± 5                                        (0.5 mL CMC oral)                                                             Intact control   222 ± 3  540 ± 22                                      (0.5 mL CMC oral)                                                             raloxifene 0.01 mg/kg, oral                                                                    176 ± 3 150 ± 5                                        raloxifene 0.10 mg/kg, oral                                                                    197 ± 3 196 ± 5                                        raloxifene 1.00 mg/kg, oral                                                                    201 ± 3 199 ± 5                                        raloxifene 10.00 mg/kg, oral                                                                   199 ± 3 186 ± 4                                        ______________________________________                                    

EXAMPLE 2

Raloxifene was administered alone or in combination with ethynylestradiol. Rats treated with raloxifene alone had uterine weights whichwere marginally higher than the ovariectomized controls and much lessthan those of ethynyl estradiol treated rats, which approached those ofthe intact controls. Conversely, raloxifene treatment significantlyreduced bone loss in ovariectomized rats, and when given in combinationwith ethynyl estradiol it did not appreciably reduce the protectiveeffect of the estrogen on bone density. The results are shown in Table4.

                  TABLE 4                                                         ______________________________________                                                       Bone Density                                                                           Uterine Weight                                                       (mg/cm/cm)                                                                             (mg)                                                  ______________________________________                                        Experiment A                                                                  Ovariectomy control                                                                            162 ± 4 142 ± 18                                       (0.5 mL CMC oral)                                                             Intact control   219 ± 5 532 ± 49                                       (0.5 mL CMC oral)                                                             EE.sub.2 100 μg/kg, oral                                                                    202 ± 6 450 ± 17                                       EE.sub.2 100 μg/kg +                                                                        204 ± 2 315 ± 10                                       raloxifene 0.10 mg/kg, oral                                                   EE.sub.2 100 μg/kg +                                                                        200 ± 5 250 ± 21                                       raloxifene 1 mg/kg, oral                                                      Experiment B                                                                  Ovariectomy control                                                                            165 ± 8 116 ± 6                                        (0.5 mL CMC oral)                                                             Intact control   220 ± 4 605 ± 69                                       (0.5 mL CMC oral)                                                             EE.sub.2 100 μg/kg, oral                                                                     215 ± 11                                                                             481 ± 24                                       raloxifene 1 mg/kg +                                                                           197 ± 7 263 ± 17                                       EE.sub.2 100 μg/kg, oral                                                   raloxifene 1 mg/kg                                                                              198 ± 11                                                                             202 ± 5                                        ______________________________________                                    

EXAMPLE 3

The ability of raloxifene to inhibit bone loss was compared to that oftamoxifen (SIGMA, St. Louis, Mo.). Tamoxifen, a well known antiestrogencurrently used in the treatment of certain cancers, has been shown toinhibit bone loss (see for example, Love, R., et al. 1992 "Effects oftamoxifen on bone mineral density in postmenopausal women with breastcancer", N Eng J Med 326:852; Turner, R., et al. 1988 "Tamoxifeninhibits osteoclast-mediated resorption of trabecular bone in ovarianhormone-deficient rats", Endo 122:1146). A relatively narrow range ofdoses of raloxifene and tamoxifen was administered orally toovariectomized rats as in the previous example. Although both of theseagents displayed the ability to prevent reduction of femur density whileevoking only modest uterotrophic activity, as identified by gains inuterine weight (Table 5), a comparison of several histologicalparameters demonstrated a marked difference between the rats treatedwith these agents (Table 6).

Increases in epithelial height are a sign of estrogenicity oftherapeutic agents and may be associated with increased incidence ofuterine cancer. When raloxifene was administered as described in Example1, only at one dose was there any statistically measurable increase inepithelial height over the ovariectomized controls. This was in contrastto the results seen with tamoxifen and estrogen. At all doses given,tamoxifen increased epithelial height equal to that of an intact rat,about a six-fold increase over the response seen with raloxifene.Estradiol treatment increased epithelial height to a thickness greaterthan intact rats.

Estrogenicity was also assessed by evaluating the adverse response ofeosinophil infiltration into the stromal layer of the uterus (Table 6).Raloxifene did not cause any increase in the number of eosinophilsobserved in the stromal layer of ovariectomized rats while tamoxifencaused a significant increase in the response. Estradiol, as expected,caused a large increase in eosinophil infiltration.

Little or no difference was detectable between raloxifene and tamoxifeneffects on thickness of the stroma and myometrium. Both agents caused anincrease in these measurements that was much less than the effect ofestrogen.

A total score of estrogenicity, which was a compilation of all fourparameters, showed that raloxifene was significantly less estrogenicthan tamoxifen.

                  TABLE 5                                                         ______________________________________                                                       Bone Density                                                                           Uterine Weight                                                       (mg/cm/cm)                                                                             (mg)                                                  ______________________________________                                        Ovariectomy control                                                                            171 ± 5 126 ± 17                                       (0.5 mL CMC oral)                                                             Intact control   208 ± 4 490 ± 6                                        (0.5 mL CMC oral)                                                             EE.sub.2 100 μg/kg, oral                                                                     212 ± 10                                                                             501 ± 37                                       raloxifene 1 mg/kg, oral                                                                        207 ± 13                                                                             198 ± 9                                        tamoxifen 1 mg/kg, oral                                                                        204 ± 7 216 ± 18                                       ______________________________________                                    

                                      TABLE 6                                     __________________________________________________________________________                Epithelial                                                                          Stromal                                                                              Myometrial                                                                          Stromal                                                    Height                                                                              Eosinophils                                                                          Thickness                                                                           Expansion                                      __________________________________________________________________________    Ovariectomy control                                                                       1.24  1.00   4.42  10.83                                          (0.5 mL CMC oral)                                                             Intact control                                                                            2.71  4.17   8.67  20.67                                          (0.5 mL CMC oral)                                                             EE.sub.2 100 μg/kg, oral                                                               3.42  5.17   8.92  21.17                                          raloxifene 1 mg/kg                                                                        1.67  1.17   5.42  14.00                                          tamoxifen 1 mg/kg                                                                         2.58  2.83   5.50  14.17                                          __________________________________________________________________________

EXAMPLE 4

Other compounds of formula I were administered orally in the rat assaydescribed in Example 1. Table 7 reports the effect of a 1 mg/kg dose ofeach compound in terms of a percent inhibition of bone loss and percentuterine weight gain.

                  TABLE 7                                                         ______________________________________                                        Compound     % Inhibition                                                                             % Uterine                                             Number       of Bone Loss.sup.a                                                                       Weight Gain.sup.b                                     ______________________________________                                        2            86         26                                                    6            24         19                                                    8            66         24                                                    10           52         24                                                    11           26         28                                                    12           60         15                                                    14           121        32                                                    16           108        25                                                    18           21         17                                                    27           25         1                                                     34           26         -6                                                    ______________________________________                                         .sup.a Percent inhibition of bone loss = (bone density of treated ovex        animals - bone density of untreated ovex animals) + (bone density of          estrogen treated ovex animals - bone density of untreated ovex animals)       × 100.                                                                  .sup.b Percent uterine weight gain = (uterine weight of treated ovex          animals - uterine weight of ovex animals) + (uterine weight of estrogen       treated ovex animals - uterine weight of ovex animals) × 100.      

EXAMPLE 5

Fracture rate as a consequence of osteoporosis is inversely correlatedwith bone mineral density. However, changes in bone density occurslowly, and are measured meaningfully only over many months or years. Itis possible, however, to demonstrate that the formula I compounds, suchas raloxifene, have positive effects on bone mineral density and boneloss by measuring various quickly responding biochemical parameters thatreflect changes in skeletal metabolism. To this end, in a current teststudy of raloxifene at least one hundred-sixty patients are enrolled andrandomized to four treatment groups: estrogen, two different doses ofraloxifene, and placebo. Patients are treated daily for eight weeks.

Blood and urine are collected before, during, and at the conclusion oftreatment. In addition, an assessment of the uterine epithelium is madeat the beginning and at the conclusion of the study. Estrogenadministration and placebo serve as the positive and negative controls,respectively.

The patients are healthy post-menopausal (surgical or natural) women,age 45-60 who would normally be considered candidates for estrogenreplacement in treatment for osteoporosis. This includes women with anintact uterus, who have had a last menstrual period more than sixmonths, but less than six years in the past.

Patients who have received any of the following medicationssystematically at the beginning of the study are excluded from thestudy: vitamin D, corticosteroids, hypolipidemics, thiazides, antigoutagents, salicylates, phenothiazines, sulfonates, tetracyclines,neomycin, and antihelmintics. Patients who have received any estrogen,progestin, or androgen treatment more recently than three months priorto the beginning of the study; patients who have ever receivedcalcitonin, fluoride, or bisphosphonate therapy; patients who havediabetes mellitus; patients who have a cancer history anytime within theprevious five years; patients with any undiagnosed or abnormal genitalbleeding; patients with active, or a history of, thromboembolicdisorders; patients who have impaired liver or kidney function; patientswho have abnormal thyroid function; patients who are poor medical orpsychiatric risks; or patients who consume an excess of alcohol or abusedrugs.

Patients in the estrogen treatment group receive 0.625 mg/day and thetwo raloxifene groups receive dosages of 200 and 600 mg/day, all groupsreceiving oral capsule formulations. Calcium carbonate, 648 mg tablets,is used as calcium supplement with all patients taking 2 tablets eachmorning during the course of the study.

The study is a double-blind design. The investigators and the patientsdo not know the treatment group to which the patient is assigned.

A baseline examination of each patient includes quantitative measurementof urinary calcium, creatinine, hydroxyproline, and pyridinolinecrosslinks. Blood samples are measured for serum levels of osteocalcin,bone-specific alkaline phosphatase, raloxifene, and raloxifenemetabolites. Baseline measurements also include examination of theuterus including uterine biopsy.

During subsequent visits to the investigating physician, measurements ofthe above parameters in response to treatment are repeated. Thebiochemical markers listed above that are associated with boneresorption have all been shown to be inhibited by the administration ofestrogen as compared to an untreated individual. Raloxifene is alsoexpected to inhibit the markers in estrogen deficient individuals as anindication that raloxifene is effective in inhibiting bone loss from thetime that treatment is begun.

Subsequent longer term studies can incorporate the direct measurement ofbone density by the use of a photon absorptiometry and the measurementof fracture rates associated with therapy.

We claim:
 1. A method of inhibiting bone loss comprising administeringto a human in need thereof an effective amount of6-hydroxy-2-(4-hydroxyphenyl)-benzo[B]thien-3-yl-4-[2-(piperidin-1-yl)ethoxyphenyl]methanonehydrochloride.
 2. The method of claim 1 wherein said human is female. 3.The method of claim 2 wherein said human has osteoporosis.